Rabies is an acute, neurological disease caused by infection of the central nervous system with rabies virus, a member of the Lyssavirus genus of the family Rhabdoviridae. Of great historical significance due to its antiquity and the horrific nature of the disease, rabies virus continues to be an important threat of human and veterinary infection because of extensive reservoirs in diverse species of wildlife. Throughout much of the world, distinct variants of rabies virus are endemic in particular terrestrial animal species, with relatively little in common between them. While several islands, including the United Kingdom, Australia, Japan, and numerous islands are free of terrestrial rabies, rabies and rabies-related viruses associated with bats have recently been identified in the UK and Australia.
Rabies virus is characteristically bullet-shaped, enveloped particle of, on average, 75 by 180 nanometers. The virion consists of a single-stranded negative sense RNA genome and five structural proteins: the nucleoprotein (N) molecules, the phospho-protein (NS), the polymerase (L), the matrix protein (M) and the viral glycoprotein (G).
The N and G proteins both bear antigenic determinants which enable serotypic characterization of diverse rabies virus strains. N determinants are highly conserved between different virus isolates and are therefore very useful targets for the immunohistological detection of rabies virus infection using specific antibodies. On the other hand, antigenic determinants carried on the G-protein vary substantially among the rabies virus strains. Virus-neutralizing antibodies raised by vaccination with inactivated virus are directed against G. While it is clear that T cell responses to G, N, and NS, participate in immune responses to the virus under experimental conditions, assessment of immunity to rabies virus is generally limited to serology, particularly with respect to virus-neutralizing antibodies.
In areas of the world where human rabies is still common, the dog is the major reservoir of the viruses that infect man. Where canine rabies has largely been eliminated by vaccination, foxes, coyotes, skunks, raccoons, bats, and a variety of other mammals harbor variants of the virus. In many areas, wildlife reservoirs of virus continue to expand. Moreover, rabies virus can be transmitted from a reservoir species to humans or other end stage hosts by animals not normally associated with rabies, such as cats, rabbits, etc.
Almost invariably fatal once clinical symptoms appear, rabies can be averted by prompt treatment of an infected individual with a combination of passive and active immunization. Passive immunization consists of the administration of pre-formed rabies virus neutralizing antibodies obtained from pooled serum of rabies immune individuals (Human rabies-immune globulin; HRIG) or hyper-immunized horses (Equine rabies-immune globulin; ERIG). Both types of reagent present certain risks to recipients including variable antigen specificity, and thus potency, for different rabies virus isolates.
HRIG is prepared from pooled human sera, therefore there is the possibility that HRIG preparations could be contaminated with known or unknown human pathogens. On the other hand, as a preparation of foreign antigen, ERIG has been associated with severe anaphylactic reactions. Mouse monoclonals specific for rabies virus have been contemplated for use in post-exposure prophylaxis but, like ERIG, are antigenically foreign to humans. This may result in their rapid clearance from the human system, as well as the potential to cause an anaphylactic reaction.
The use of human monoclonal antibodies is limited since human hybridoma cell lines are hard to prepare, generally unstable, and do not produce monoclonal antibodies of appropriate specificity in sufficient quantities and at reasonable costs. Production costs of monoclonal antibodies make it desirable to find more economic alternatives to obtaining monoclonal antibodies from hybridomas.
It is well established that both the Fab and Fab2 regions, which comprise the variable and hinge regions of the heavy and light chains, do not protect against rabies virus infection. The in vivo efficacy of the antibody relies on the entire sequence, that is only particular antibodies exhibit anti-rabies activity. It is the constant region of the antibody that is responsible for immunoreactivity. Thus, it is particular attributes of the constant region(s) that are required to protect against the rabies virus. Variable regions spliced to a constant region of another antibody, that is an antibody that is not naturally made against the rabies virus, are ineffective.
There is a need for recombinant antibodies useful in the diagnosis, prevention and treatment of rabies infection, and pharmaceutical compositions that comprise and methods that use the same. There is a need for compositions and methods for producing such recombinant antibodies.
There is a need for recombinant antibodies useful in the diagnosis, prevention and treatment of infection of pathogens that target neuronal tissue, and pharmaceutical compositions that comprise and methods that use the same. There is a need for compositions and methods for producing recombinant antibodies.
To provide a better reagent, human monoclonal antibodies have been made by fusion of Epstein-Barr Virus (EBV)-transformed, rabies virus-specific human B cells with mouse-human heterohybrid donors. cDNA clones encoding the antibody heavy and light chains from these cells were constructed such that the antibodies were expressed in heterologous expression systems. These constructs allow rabies neutralizing human antibodies of defined specificity to be produced in a controlled system, purified away from possible deleterious contaminants. The present invention relates to these monoclonal rabies virus neutralizing human antibodies, the nucleic acid sequences of their heavy and light chains and the amino acid sequences of the encoded proteins. Also provided in the present invention are methods of using the monoclonal antibodies as a therapeutically effective post-exposure prophylactic treatment of individuals exposed to rabies virus.